We developed a genetic mapping resource which allows efficient construction of genetic maps in three dimensions. In collaboration with leading comparative gene mappers in mice and cattle, we developed 318 polymerase chain reaction (PCR)-based comparative mapping anchor tagged sequences (CATS) for the construction of Type I (coding gene) comparative genetic maps. This resource can be used to efficiently determine order and distance of genes within any vertebrate species and it allows accurate comparisons across species. The CATS represent coding genes which have been previously mapped by recombination analyses in the mouse and by physical methods in humans. The CATS are evenly spaced at 5 - 10 cM intervals and sequenced PCR products showed that the CATS amplify the correct genes. The primers for the CATS proved to be well conserved across vertebrate species and can be used by any vertebrate mapping project. Single stranded conformation polymorphism (SSCP) screening has been conducted of CATS products which generate a single PCR product and whose sequence matches its human or mouse homologue in GenBank. Type I markers which exhibited polymorphism between the parent species of the interspecific backcross pedigree (the domestic cat and Asian leopard cat) were subsequently genotyped in the 118-member three-generation interspecies backcross pedigree developed for generation of the feline linkage map. Additionally, primers designed in 3? untranslated regions of either feline-specific expressed sequence tags (237) or feline genes deposited in GenBANK (71) and feline locus-specific sequence tagged sites (35) were amplified in the interspecies pedigree and examined for polymorphism (SSCP). Approximately 33% of PCR products exhibited a polymorphism between the domestic cat and leopard cat and 100 of these Type I loci generated interpretable genotype patterns in the interspecies pedigree. Genotype analysis is currently in progress. Recently a linkage map of 253 microsatellites was generated in the interspecies pedigree as a collaborative effort with Alejandro Schaffer at NHGRI/DRB. Linkage groups in the microsatellite map were physically assigned to all of the 18 feline autosomes and the X chromosome using a cat x rodent somatic cell hybrid panel. Microsatellite average locus heterozygosity of 0.74 was observed. An average locus heterozygosity over the marker set of 0.68 observed in Egyptian mau, a domestic cat breed that contributed to part of the pedigree, suggest that the marker set will be adequately polymorphic within cat breeds to be informative in gene mapping exercises within cat breeds that segregate for inherited disease pathology modeling human hereditary conditions. The map had an average intragroup intermarker spacing of 11 cM and genome coverage spanned aproximately 2900 cM. The 100 Type I markers will be integrated with the existing microsatellite linkage map to provide a valuable resource for mapping phenotypic variationin the species and relating it to gene maps of other mammals, including human. Additionally, the feline microsatellites have proved to be a reliable resource for genetic individualization in conservation biology and forensic applications. - backcross, cat, comparative, feline, forensic, interspecific, microsatellite, painting, coding genes,